An oligonucleotide binds to a target nucleic acid by forming hydrogen bonds between bases in the target and the oligonucleotide. Common B DNA has conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watson and Crick base pairs with two and three hydrogen bonds, respectively. Conventional hybridization technology is based upon the capability of sequence-specific DNA or RNA probes to bind to a complementary target nucleic acid via Watson-Crick hydrogen bonds. However, other types of internucleotide hydrogen bonding patterns are known wherein atoms not involved in Watson-Crick base pairing to a first nucleotide can form hydrogen bonds to another nucleotide. For example, thymine (T) can bind to an AT Watson-Crick base pair via hydrogen bonds to the adenine, thereby forming a T-AT base triad. Hoogsteen (1959, Acta Crystallographica 12:822) first described the alternate hydrogen bonds present in T-AT and C-GC base triads. More recently, G-TA base triads, wherein guanine can hydrogen bond with a central thymine, have been observed (Griffin et al., 1989, Science 245:967-971).
Oligonucleotides which can bind to a target with both Watson-Crick and non-Watson-Crick hydrogen bonds form extremely stable complexes which have a variety of in vivo and in vitro utilities. To date there has been no disclosure of a stem-loop oligonucleotide with the necessary structural features to achieve stable target binding via Watson-Crick and alternate hydrogen bonds.
Oligonucleotides have been used for a variety of utilities. For example, oligonucleotides can be used as probes for target nucleic acids that are immobilized onto a filter or membrane, or are present in tissues, e.g. as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, New York). However, the utility of linear oligonucleotide probes is frequently limited by their poor binding stability and selectivity.
Furthermore, there has been great interest recently in developing oligonucleotides as regulators of cellular nucleic acid biological function. This interest arises from observations on naturally occurring complementary, or antisense, RNA used by some cells to control protein expression. However, the development of oligonucleotides for in vivo regulation of biological processes has been hampered by several long-standing problems, including the low binding affinity and nuclease sensitivity of linear oligonucleotides.
For example, transcription of the human c-myc gene has been inhibited in a cell free, in vitro assay system by a 27-base linear oligonucleotide designed to bind to the c-myc promoter (Cooney et al., 1988, Science 241:456). Inhibition was only observed using a carefully controlled in vitro assay system wherein lower than physiological temperatures were employed, and many cellular enzymes had been removed or inactivated. These conditions were necessary because linear oligonucleotides bind with low affinity and are highly susceptible to enzymes which degrade linear pieces of DNA.
Splicing of a pre-mRNA transcript essential for Herpes Simplex virus replication has also been inhibited with a linear oligonucleotide which was complementary to an acceptor splice junction. In this instance, a methylphosphonate linkage was employed in the linear oligonucleotide to increase its nuclease resistance. Addition of this chemically-modified oligonucleotide to the growth medium caused reduction in protein synthesis and growth of uninfected cells, most likely because of toxicity problems occurring at high oligonucleotide concentrations (Smith et al., 1986, Proc. Natl. Acad. Sci. USA 83:2787-2791).
In another example, linear oligonucleotides were used to inhibit human immunodeficiency virus replication in cultured celts. Linear oligonucleotides complementary to sites within or near the terminal repeats of the retrovirus genome and within sites complementary to certain splice junctions were most effective in blocking viral replication. However, these experiments required large amounts of the linear oligonucleotides before an effect was obtained, presumably because of the low binding affinity and vulnerability of these linear oligonucleotides to nucleases (Goodchild et al., 1988, Proc. Natl. Acad. Sci. USA 85:5507-5511).
Linear oligonucleotides have been observed to bind by non-Watson-Crick or Watson-Crick hydrogen bonding in vitro. However, linear oligonucleotides which are bound to target via only non-Watson-Crick or only Watson-Crick hydrogen bonds are not very stable, and can be readily displaced from the target under normal cellular conditions.
Xodo et al. (1990, Nucleic Acids Res. 18:3557-3564) and Giovannangeli et al. (1991, J. Am. Chem. Soc. 113:7775-7777) disclose linear pyrimidine oligonucleotides in which one domain of the oligonucleotide reportedly forms an antiparallel duplex with a target purine sequence, and a second domain "folds back" and binds to the duplex, thus forming a bimolecular triple helix. The resulting "U-shaped" oligonucleotides of Xodo et al. and Giovannangeli et al. thus apparently bind the target with both Watson-Crick and non-Watson-Crick bonds. However, it has been found in accordance with the present invention that such linear oligonucleotides do not bind with optimal affinity.
Baumann et al. (1988, Blochem. Biophys. Res. Commun. 157:986-991) disclose stem-loop oligonucleotides wherein the loop can intermolecularly bind to a single stranded oligomer by Watson-Crick base pairing only. This interaction appears to mimic the native interaction that occurs between the tRNA anticodon loop and the mRNA codon.
Single-stranded circles of DNA or RNA are known which can bind to target via both Watson-Crick and non-Watson-Crick hydrogen bonding (Kool, 1991, J. Amer. Chem. Soc. 113:6265-6266; and Prakash et al., 1991, J. Chem. Soc. Chem. Commun. 113:1161-1163). However, Kool and Prakash et al. are particularly drawn to circular oligonucleotides and do not disclose or teach stem-loop oligonucleotides.
Moreover, such circular oligonucleotides are not always ideal for in vivo regulation of biosynthetic processes. For example, such circular oligonucleotides can be made only in vitro. In contrast, the present invention provides stem-loop oligonucleotides which are easily synthesized by recombinant technology in vivo. Furthermore, the in vitro synthesis of circular oligonucleotides can be expensive and time-consuming relative to the easily synthesized oligonucleotides of the present invention.
The present invention provides stem-loop oligonucleotides which have many of the desirable attributes of circular oligonucleotides, e.g. nuclease resistance (Tang et al., 1993, Nucleic Acids Res. 21:2729-2735). However the present stem-loop oligonucleotides are much simpler to make both in vivo and in vitro. Moreover, the present stem-loop oligonucleotides bind target via both Watson-Crick and non-Watson-Crick hydrogen bonding. The present stem-loop oligonucleotides bind with strong affinity and high selectivity to their targeted nucleic acids.